We are analyzing the interactions between virus glycoproteins and their receptors for several enveloped viruses of public health significance in the US and worldwide. Our goals are to define mechanisms of virus entry and cell tropism, identify cellular receptors that mediate virus entry, and develop novel antiviral approaches for treatment (antiviral drugs) and prevention (vaccines, microbicides). 1) Kapsosi's Sarcoma-Associated Virus (KSHV, aka human herpesvirus 8). Our previous work demonstrated that a broad range of human and non-human primate cell types can serve as efficient targets in assays of KSHV glycoprotein-mediated cell fusion and KSHV virion entry. We identified a novel KSHV receptor: xCT, the 12 transmembrane light chain of the xc- cystine/glutamate exchange transport system. During the past year, we focused on obtaining reagents for structure-function studies of the KSHV interaction. To this end, we obtained monoclonal antibodies against xCT from Dr. D. Afar, Protein Design Labs. Two were found to inhibit KSHV cell fusion; curiously, these Mabs did not affect KSHV infection. We have also prepared mg quantities of soluble constructs of KSHV glycoproteins involved in fusion (gH and gB); these will be valuable probes to study gp-receptor interactions. We have also begun collaborative studies with Dr. Leonid Margolis, NICHD, to study KSHV infection in lymphoid tissue explants, with the goals of defining the role of xCT and determining whether this receptor is used for infection of B cells. In a new collaborative effort with Dr. Corey Casper and Dr. Larry Corey, FHCRC, U. Washington, we plan to develop immunotoxins against KSHV proteins for treatment of diseases associated with KSHV infection. For Multicentric Castlemans Disease in which most cells are in the lytic phase, we will make immunotoxins targeting glycoproteins gH, gB, and K8.1A, using previously described or newly generated hybridomas. Approaches will include murine systems (spleen cell fusions, cDNA phage libraries) as well as infected human sources (cDNA phage libraries). For Kaposis sarcoma, we will target the viral G protein coupled receptor which has been implicated in initiation and maintenance of KS tumors. We have initiated a collaboration with Dr. Silvio Gutkind, NIDCR, to produce variant constructs of the vGPCR that yield high surface expression levels; this will facilitate elicitation and analysis of monoclonal antibodies to be used for immunotoxin production. Dr. Ira Pastan, a collaborator in our HIV immunotoxin work, has offered assistance with this effort (though not a formal collaborator). Dr. Robert Purcell has also expressed interest in phage display systems for isolating the desired antibodies. 2) Hepatitis C virus (HCV). We are particularly interested in developing systems to study HCV entry, for use in defining essential receptors and elucidating entry mechanisms, assaying HCV neutralizing antibodies in infected people and in vaccine studies. To this end, we are devising systems to produce HCV virus-like particles containing the functional HCV E1 and E2 glycoproteins and containing an RNA that encodes a reporter gene; this will enable rapid, quantitative, and sensitive measurement of entry. During the past year, we have had considerable success with a novel system in which a full length HCV genome encoding the structural genes but not the nonstructural genes, is introduced into a particular cell line (containing a flavivirus replicon) that produces and secretes infectious (single cycle) HCV virus-like particles. The genome also encodes a component of a novel reporter system based on expression of T7 RNA polymerase from a single RNA molecule introduced by an HCV VLP. We have obtained convincing evidence for VLP entry mediated by the viral E1?E2 glycoproteins, and dependent on previously defined HCV receptors (CD81, asialoglycoprotein receptor, SRBP).